Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis
Tomohiro Watanabe, Naoki Asano, Peter J. Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J. Fuss, Atsushi Kitani, Warren Strober
Tomohiro Watanabe, Naoki Asano, Peter J. Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J. Fuss, Atsushi Kitani, Warren Strober
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Research Article Gastroenterology

Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis

Abstract

The mechanisms underlying the susceptibility of individuals with caspase recruitment domain 15 (CARD15) mutations and corresponding abnormalities of nucleotide-binding oligomerization domain 2 (NOD2) protein to Crohn disease are still poorly understood. One possibility is based on previous studies showing that muramyl dipeptide (MDP) activation of NOD2 negatively regulates TLR2 responses and that absence of such regulation leads to heightened Th1 responses. We now report that administration of MDP protects mice from the development of experimental colitis by downregulating multiple TLR responses, not just TLR2. The basis of these in vivo findings was suggested by in vitro studies of DCs, in which we showed that prestimulation of cells with MDP reduces cytokine responses to multiple TLR ligands and this reduction is dependent on enhanced IFN regulatory factor 4 (IRF4) activity. Further studies of mouse models of colitis showed that this inhibitory role of IRF4 does in fact apply to MDP-mediated protection from colitis, since neither IRF4-deficient mice nor mice treated with siRNA specific for IRF4 were protected. These findings indicate that MDP activation of NOD2 regulates innate responses to intestinal microflora by downregulating multiple TLR responses and suggest that the absence of such regulation leads to increased susceptibility to Crohn disease.

Authors

Tomohiro Watanabe, Naoki Asano, Peter J. Murray, Keiko Ozato, Prafullakumar Tailor, Ivan J. Fuss, Atsushi Kitani, Warren Strober

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Figure 6

Human and mouse DCs prestimulated with MDP exhibit reduced cytokine and chemokine production when stimulated with TLR ligands.

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Human and mouse DCs prestimulated with MDP exhibit reduced cytokine and ...
(A) Human monocyte–derived DCs (1 × 106/ml) from 6 healthy donors were preincubated with MDP or medium for 24 hours and then stimulated with a broad range of TLR ligands alone or in combination with MDP for an additional 24 hours. Cultured supernatants were collected at 24 hours and analyzed for cytokine and chemokine production by ELISA. *P < 0.05; **P < 0.01 compared with supernatants from DCs preincubated with medium and stimulated with TLR ligands alone (light blue bars). (B) CD11c+ DCs (1 × 106/ml) derived from bone marrow cells from NOD2-intact (NOD2+/+) and NOD2-deficient (NOD2–/–) mice were preincubated with MDP (50 μg/ml) or medium alone for 24 hours and stimulated with a broad range of TLR ligands. Cultured supernatants were collected at 24 hours and analyzed for cytokine production by ELISA. *P < 0.05; **P < 0.01 when supernatants were compared with NOD2-intact DCs preincubated with medium and stimulated with TLR ligands (light blue bars). (C) OVA323-339 peptide–specific CD4+ T cells (OT-II) were purified from the spleens of OT-II transgenic mice; OT-II cells (1 × 106/ml) were cocultured with NOD2-intact or NOD2-deficient BMDCs (2 × 106/ml) in the presence of a broad range of TLR ligands and OVA peptide (0.5 μM); cultured supernatants were collected at 72 hours and analyzed for IFN-γ production by ELISA. *P < 0.05; **P < 0.01 compared with supernatants from NOD2-intact DCs preincubated with medium and stimulated with TLR ligands (light blue bars).